Over the past decade, the study of how G protein-coupled receptors (GPCRs) control cell growth, proliferation and differentiation has fundamentally changed our view of GPCR signal transduction. Far from the canonical model in which GPCRs function solely as activators of heterotrimeric G proteins, we now recognize that they are versatile signaling platforms that transmit both G protein-dependent and -independent signals. Our research originally focused on GPCR regulation of the ERK1/2 MAP kinase cascade. We established that GPCRs use several mechanistically distinct pathways to control ERK1/2 activity, including G protein- dependent signals transmitted by second messenger-dependent protein kinases and 'transactivated'EGF receptors, and novel G protein-independent signals that result from the -arrestin-dependent assembly of multiprotein 'signalsomes'. These results have defined two distinct GPCR signaling 'modes', and in some cases we have identified pathway-selective 'biased agonists'that dissociate them. Moreover, we have found that these pathways are not functionally redundant. Rather, the mechanism of activation determines the time course, spatial distribution, and ultimately the function of GPCR-regulated kinases. The central hypothesis of this proposal is that heterotrimeric G proteins and -arrestins serve as independent GPCR signal transducers that mediate distinct facets of the cellular response to GPCR stimulation. The proposal is organized into three Specific Aims, the first two focused on the structure and function of the GPCR-arrestin 'signalsome'and the third on how G protein-dependent and -arrestin-dependent signals are integrated to determine the cellular response. In each aim, we will focus on the angiotensin AT1A receptor, which utilizes both signaling mechanisms. Aims I and II employ transfected cell systems that allow us to use receptor and -arrestin mutants and rapid siRNA silencing of protein expression to maximum advantage. Experiments will determine the composition of the AT1AR--arrestin 'signalsome'and the structural features of the receptor and -arrestin that dictate signalsome composition and stability. We will employ advanced proteomic methodology to determine how G protein-independent signaling affects protein phosphorylation and determine how -arrestin signaling affects gene transcription. Aim III will concentrate on signaling by endogenous AT1A receptors in primary aortic vascular smooth muscle cells. We will employ pathway-selective agonists, pharmacologic inhibitors and shRNA expression silencing to study the cellular processes regulated by each type of signal in a physiologically relevant context. Experiments will determine the temporal, spatial and functional characteristics of the different types of signal, and how they are integrated to produce cellular changes associated with the development of atherosclerotic vascular disease. Completion of these studies will address a fundamental gap in our understanding of how GPCRs work and may provide insights into novel therapeutic applications of GPCR ligands with pathway-selective agonist or antagonist properties. PUBLIC HEALTH RELEVANCE: Contrary to the traditional view that G protein-coupled receptors (GPCRs) only signal by activating heterotrimeric G proteins, recent research has shown that they also transmit G protein-independent signals that are initiated by binding to adapter or scaffold proteins. This project focuses on the role of arrestins in angiotensin AT1A receptor signaling. Arrestins bind to activated GPCRs, 'uncoupling'them from G proteins while at the same time promoting the assembly of 'signalsomes'that affect protein phosphorylation and gene transcription. Our research will define the factors that control the assembly and function of the AT1A receptor- arrestin signalsome and determine how G protein-dependent and arrestin-dependent signals are integrated. These studies address fundamental gaps in our understanding of how GPCRs work and may provide insights into novel therapeutic applications of GPCR ligands with pathway-selective agonist or antagonist properties.